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. 2022 Apr 26;34(5):2001-2018.
doi: 10.1093/plcell/koac026.

The tomato OST1-VOZ1 module regulates drought-mediated flowering

Affiliations

The tomato OST1-VOZ1 module regulates drought-mediated flowering

Leelyn Chong et al. Plant Cell. .

Abstract

Flowering is a critical agricultural trait that substantially affects tomato fruit yield. Although drought stress influences flowering time, the molecular mechanism underlying drought-regulated flowering in tomato remains elusive. In this study, we demonstrated that loss of function of tomato OPEN STOMATA 1 (SlOST1), a protein kinase essential for abscisic acid (ABA) signaling and abiotic stress responses, lowers the tolerance of tomato plants to drought stress. slost1 mutants also exhibited a late flowering phenotype under both normal and drought stress conditions. We also established that SlOST1 directly interacts with and phosphorylates the NAC (NAM, ATAF and CUC)-type transcription factor VASCULAR PLANT ONE-ZINC FINGER 1 (SlVOZ1), at residue serine 67, thereby enhancing its stability and nuclear translocation in an ABA-dependent manner. Moreover, we uncovered several SlVOZ1 binding motifs from DNA affinity purification sequencing analyses and revealed that SlVOZ1 can directly bind to the promoter of the major flowering-integrator gene SINGLE FLOWER TRUSS to promote tomato flowering transition in response to drought. Collectively, our data uncover the essential role of the SlOST1-SlVOZ1 module in regulating flowering in response to drought stress in tomato and offer insights into a novel strategy to balance drought stress response and flowering.

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Figures

Figure 1
Figure 1
OST1 regulates flowering transition in tomato. A, Phylogenetic analysis of SnRK2 proteins from Arabidopsis and tomato, generated in MEGA 7 with the neighbor-joining method. B, Protein sequence alignment of SlOST1 and AtOST1. Red asterisks indicate the conserved amino acids required for OST1 kinase activity. C, Schematic diagram of the two sgRNAs designed to specifically edit SlOST1 in two tomato genetic backgrounds, MT and AC, by CRISPR–Cas9. D, Delayed flowering of slost1 mutants compared to WT plants (MT) under LD conditions. Data represent mean ± standard deviation (sd, n = 6). E, Late flowering of slost1 mutants compared to WT plants (AC) under LD and SD conditions. Data represent mean ± sd (n = 6). *P < 0.05, **P < 0.01, Student’s t test relative to WT. Scale bars, 5 cm.
Figure 2
Figure 2
SlOST1 physically interacts with and phosphorylates SlVOZ1. A, SlOST1 phosphorylates SlVOZ1 in vitro. Recombinant purified GST-SlOST1 was incubated with GST-SlVOZ1 or GST-SlVOZ1S67A in kinase reaction buffer. The proteins were separated by SDS–PAGE. Top, autoradiogram; bottom, Coomassie Brilliant Blue staining. B, Phosphorylation of SlVOZ1 by SlOST1 in transfected Arabidopsis protoplasts. SlVOZ1-MYC was co-transfected alone or with increasing amounts of SlOST1-HA plasmid DNA in protoplasts. Protein abundance and mobility shift were detected by immunoblot and Phos-Tag assay, respectively. The ratio of p-SlVOZ1 to SlVOZ1 is shown. C, LCI assay showing the physical interaction between SlOST1 and SlVOZ1. D, Co-IP assay demonstrating the interaction between SlOST1 and SlVOZ1 in protoplasts. SlOST1-HA was precipitated with anti-HA agarose. SlOST1 and SlVOZ1 proteins were separated by immunoblot and detected with anti-HA and anti-MYC antibodies, respectively. E, BiFC assay in N. benthamiana leaves showing the specific interaction between SlOST1 and SlVOZ1. Schematic diagrams indicate the various constructs encoding full-length or truncated SlVOZ1 fused to cYFP; SlOST1 was fused to nYFP. H2B-mcherry was co-infiltrated as a nuclear marker. Scale bars, 50 µm.
Figure 3
Figure 3
SlOST1 promotes the protein stability of SlVOZ1. A, In vitro cell-free degradation assay showing the effects of SlOST1 on SlVOZ1 and SlVOZ1S67A degradation. Equal amounts of recombinant GST-SlOST1, GST-SlVOZ1, or GST-SlVOZ1S67A proteins were incubated with total protein extracts from WT tomato seedlings for 15, 30, and 45 min without or with 10-µM MG132 added. SlOST1 and SlVOZ1 proteins were detected with anti-GST antibody. Actin served as a loading control. B, SlVOZ1 protein stability is enhanced by SlOST1 in protoplasts. An equal amount of SlVOZ1-MYC, SlVOZ1S67A-MYC, and SlOST1-HA (in an increasing concentration) plasmid DNA was co-transfected in protoplasts. SlOST1, SlVOZ1, and SlVOZ1S67A-MYC protein abundance as determined by immunoblot. Actin served as a loading control. C, SlVOZ1 protein abundance and SlVOZ1 transcript levels in WT and slost1 mutants. Total proteins from different tissues were extracted from 9-week-old tomato plants and were probed with anti-SlVOZ1 antibody. Protein levels were quantified using ImageJ. The gene expression data were normalized to WT and represent means ± sd from three biological replicates. D, ABA enhances the protein stability and phosphorylation of SlVOZ1. The SlVOZ1 construct was transfected alone or with SlOST1 in protoplasts treated with ABA. The protein stability and phosphorylation of SlVOZ1 were determined by immunoblot and Phos-Tag, respectively. λPPase treatment was applied to abolish the phosphorylation of SlVOZ1.
Figure 4
Figure 4
SlOST1 regulates the nuclear translocation of SlVOZ1. A, Representative images showing the subcellular localization of SlVOZ1 with or without ABA and CHX treatments. Four-day-old SlVOZ1-GFP transgenic Arabidopsis seedlings were treated with liquid MS medium without or with ABA and CHX before observing GFP fluorescence by confocal microscopy. The GFP intensity in the selected root region was quantified by ZEN version 3.2 software. Scale bars, 50 µm. B, Immunoblot results showing the nuclear accumulation of SlVOZ1 after ABA treatment. Seven-day-old SlVOZ1-GFP transgenic Arabidopsis seedlings were treated with 50-µM ABA for 3 h before harvest. The accumulation of SlVOZ1 and nuclear proteins were detected by anti-SlVOZ1 and anti-H3 antibodies, respectively. C, Immunoblot assay showing the accumulation of SlVOZ1 in WT and slost1 mutants. T, total protein; C, cytosolic protein; and N, nuclear proteins isolated from 7-day-old tomato seedlings using the CelLytic PN extraction kit. The abundance of SlVOZ1 was detected with anti-SlVOZ1 antibody; cytosolic and nuclear proteins were detected with anti-HSP70 and anti-H3 antibodies, respectively.
Figure 5
Figure 5
SlVOZ1 is essential for the flowering transition in tomato. A, Schematic diagrams of the sgRNA designed to target SlVOZ1 with CRISPR–Cas9 and independent CRISPR alleles created in the MT and AC backgrounds. B, Flowering phenotype in WT and slvoz1 mutants (MT background) under LD conditions. Data represent mean ± sd (n = 6). C, The slvoz1 mutants are late flowering (AC background), as determined by leaf number under LD and SD conditions. Data represent mean ± sd (n = 6). **P < 0.01, Student’s t test, relative to WT. Scale bars, 5 cm.
Figure 6
Figure 6
Drought-accelerated flowering is partially dependent on the SlOST1–SlVOZ1 module. A and B, Representative phenotypes and electrolyte leakage of WT, slost1 and slvoz1 mutants (MT background) under control (H2O) and 200-mM mannitol treatment conditions. Data are means ± sd of three biological replicates. Different letters indicate significant differences by two-way ANOVA (Analysis of Variance) (Duncan’s multiple range test, P < 0.05). C, WLR of WT, slost1 and slvoz1 mutants. Data are means ± sd of three biological replicates. D, False-colored infrared-thermal images of the WT, slvoz1 and slost1 mutant plants. E, Flowering phenotype of WT, slost1 and slvoz1 mutants (MT background) under normal and drought treatment conditions. Scale bars, 5 cm. F, Flowering time of WT, slost1 and slvoz1 mutants under normal and drought treatment conditions. Data represent mean ± sd (n = 6), and experiments were repeated 3 times independently with similar results. G, Relative SFT transcript levels in 18-day-old WT, slost1, slvoz1 seedlings under control and mannitol treatment conditions. Tomato Actin 7 was used as a reference control. Data represent mean ± sd from three technical replicates. Different letters represent significant differences, as determined using two-way ANOVA with Tukey’s post hoc test (P < 0.05).
Figure 7
Figure 7
Genome-wide identification of SlVOZ1 binding motifs and target genes by DAP-seq. A, Venn diagram showing the extent of overlap between peaks identified from the two DAP-seq replicates. B, Distribution of high-confidence SlVOZ1 binding peaks along the tomato genome. C, GO enrichment analysis of SlVOZ1 binding peaks. D, The five enriched binding motifs identified for SlVOZ1. E, Validation of the direct binding of SlVOZ1 to the five enriched motifs by EMSA. FAM-labeled DNA probes were incubated with recombinant GST-SlVOZ1 or GST-SlOST1 proteins.
Figure 8
Figure 8
SlVOZ1 directly binds to the SFT promoter to activate transcription. A, Illustration of the binding motifs of SlVOZ1 in the SFT promoter. B, EMSA showing the direct binding of SlVOZ1 to the SFT promoter probes. Recombinant GST-SlVOZ1, GST-SlOST1, or both proteins were incubated with FAM-labeled DNA fragments. C, Schematic diagrams of the effector and reporter constructs used for the transactivation assay. D, Transactivation assay showing that SlVOZ1 activates the transcription of SFT. **P < 0.01, Student’s t test, relative to WT. The protein abundance of effectors was detected by immunoblot using anti-GFP antibody. E, ChIP-qPCR assay showing the binding of SlVOZ1 to the SFT promoter in vivo. Leaves of 6-week-old tomato plants grown under LD conditions were harvested in the morning for ChIP. Data represent means ± sd of three technical repeats. *P < 0.05, **P < 0.01, Student’s t test, relative to WT. Anti-SlVOZ1 antibody was used to precipitate the SlVOZ1–DNA complex in WT and slvoz1 mutants. ChIP experiments were performed two independent times with similar results.
Figure 9
Figure 9
Model illustrating how the SlOST1–SlVOZ1 module balances flowering transition and drought. Upon encountering drought stress, SlOST1 is activated to interact with and phosphorylate SlVOZ1, which leads to enhanced protein stability and nuclear accumulation of SlVOZ1. Nucleus-localized SlVOZ1 then binds to the SFT promoter to promote flowering transition in response to drought stress.

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